The tightly-coordinated activities of proteases are important in normal cellular functions. However, dysregulation of proteolysis has been linked to cancer progression and metastasis. The type II transmembrane serine proteases (TTSPs) under investigation in the research plan are a family of surface-expressed proteases that are upregulated in many epithelial-derived cancers. Although TTSP-mediated proteolysis is known to activate pro-cancer pathways, essential details regarding TTSP activity and regulation are unknown. The extracellular localization of TTSPs makes them ideal targets for cancer diagnosis and therapeutic applications. However, much progress needs to be made in assessing the molecular details of TTSP function throughout tumor development. Dissecting the roles of TTSP activity in cancer requires chemical tools that can specifically target unique members of the TTSP family. Although probes have been successfully developed to monitor the activity of other proteases in vivo, many activity-based probes do not provide a complete picture of protease activity with high spatial and temporal resolution. Substrate-based probes that are activated upon cleavage have allowed for the real-time monitoring of proteolysis in vivo but designing unique substrates for members of the same protease family can be challenging. In the research plan, protease-specific probes for two members of the TTSP family, MT-SP1 and TMPRSS2, are designed and implemented in order to better assess the functional roles of the proteases in prostate cancer. A strategy for building peptide libraries to profile protease cleavage specificities is introduced that maximizes physiochemical diversity within a small amino acid sequence space. Using this generalizable approach, novel TTSP-specific probes with a turn-on fluorescence response to proteolysis will be generated based on non-overlapping peptide cleavage sites. The specificity of the probes will be evaluated in vitro and in a prostate cancer cell line using a high-throughput, continuous assay. The probes that are found to be the most selective for their intended target will be used to simultaneously image endogenous MT-SP1 and TMPRSS2 activity in real-time using preclinical animal models for prostate cancer. This line of investigation will better define the roles of MT-SP1 and TMPRSS2 in cancer development as well as provide a dynamic profile of MT- SP1 and TMPRSS2 activity that could inform new cancer treatment strategies.